T hesc

Human hTERT-immortalized endometrial stromal fibroblasts were purchased from ATCC (T-HESC; CRL-4003, ATCC). Their identity has been authenticated by ATCC, and was determined to be mycoplasma free.

Telomerase-immortalized human endometrial stromal cells (T-HESC, ATCC CRL-4003) [31 (link)] were cultured in DMEM/Nutrient Mixture Ham's F-12 with l-glutamine (Sigma Aldrich, St. Louis, MO), 1% BD ITS+ Universal Culture Supplement Premix (BD Biosciences, San Jose, CA), 1.5 g/liter sodium bicarbonate, 2% penicillin / streptomycin, and 10% charcoal-treated FBS (HyClone), which is referred to as HESC medium herein. For the cell experiments, T-HESCs were decidualized (dT-HESCs) as described [19 (link), 20 (link)] by incubating the cells with 0.5 mM 8-bromo-cyclic amp (cAMP) (Sigma Aldrich) for three to six days. Completely confluent monolayers were used for all experiments to ensure that only decidual cell surfaces were available for bacterial interactions.

Human endometrial stromal cells (T-HESC; ATCC No. CRL-4003) [34 (link)] were cultured in phenol red-free DMEM/F12 supplemented with 10% charcoal-stripped fetal bovine serum (FBS; HyClone), 1% ITS + Premix (BD), 500 ng/ml puromycin (Invitrogen) and 100 U/ml Penicillin and 100 μg/ml Streptomycin. Cells were maintained at 37°C supplemented with 5% CO2. Authentication of cells was performed by ATCC using short tandem repeat (STR) analysis. The cells were further verified using a functional assay to demonstrate their ability to undergo decidualization response via increasing the expression of insulin like growth factor binding protein 1 (IGFBP1) mRNA upon treatment with 8-bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cAMP) (0.5 mM) (S1 Fig). For TGFB1 treatment, the cells were serum-starved overnight and then treated with vehicle (VEHL) or TGFB1 (0.1–10 ng/ml; R&D) for 24 h. Cells were collected and total RNA and proteins isolated as described below.

Human pancreatic adenocarcinoma cells (PDAC) such as BxPC-3 and AsPC-1, and immortalized human normal endometrial fibroblast cells (T-HESC) were purchased from American Type Culture Collection (VA, USA). Human pancreatic stellate cells (PSC) were isolated from the resected pancreas tissues of patients undergoing operation for pancreatic cancer as previously described [53 (link)], under the approval by the Ethics Committee of Tohoku University School of Medicine (article#: 2009-79 and 2014-1-132), while human endometrial adenocarcinoma primary fibroblast cells (EC6/Fib) were isolated from resected endometrium tissues [54 (link)]. PDAC and EC6/Fib were maintained in RPMI-1640, while PSC were maintained in DMEM/F-12 media, all supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Life Technologies) at 37°C in 95% humidified atmosphere of 5% CO₂.